Abstract Lung transplant is the only definitive therapy available for several devastating lung diseases including emphysema/COPD, cystic fibrosis, and interstitial lung disease. However, the current utility of lung transplant is hampered a shortage of lung donors, the propensity for lungs to become infected, and rejection of the transplanted organ. Whole organ bioengineering offers a potential solution to these problems by removing all immunogenic and infectious material from donated human or animal lungs, and seeding the remaining scaffold with cells derived from the proposed recipient. This decell/recell strategy has been investigated in many different organs across many different labs, but due to the unique nature of the lung (the fragile alveoli and air- filled bronchi), techniques developed for other organs are not well-suited for the lung. Many of these techniques circulate decellularization reagents using fairly high pressures which may damage the delicate alveolar structure similar to the clinical entity known as ventilator associated lung injury. These techniques also rely on aqueous cell suspensions for recellularization. This poses numerous difficulties in the airways. Due to the size, shape, and single opening of airways, cells settle to the dependent areas and become trapped in the small airways rather than adhering to the large airways. Additionally, bronchial cells require exposure to an air liquid interface to properly proliferate and mature. Finally, there is growing evidence suggesting that airflow patterns within the airways are important determinants of lung health but few techniques exist to investigate these flow patterns and it is not known what influence decellularization, or transplant will have on these airflows let alone the impact of the anastomosing animal lungs to human airways in xenotransplant. In this proposal, I aim to investigate new decellularization and recellularization techniques which better approximate normal physiology. This will be accomplished through three specific aims 1) Determine if negative pressure ventilation is less injurious to pulmonary tissue during decellularization and if gas ventilation improves cell proliferation and maturation during recellularization. 2) Determine optimal characteristics of a novel cell sprayer device to apply cells to the airways. 3) Evaluate a new technique to assess airflow patterns within the airways.